Circadian clocks are used by almost all organisms use to keep time. Circadian clocks control a wide variety of fundamental cellular, physiological, and behavioral processes. The molecular machinery that permits the measurement of time is referred to as the biological clock or circadian clock and its output as circadian rhythms. Our long-term goal is to understand the molecular and biochemical mechanisms of circadian clocks. The filamentous fungus Neurospora crassa, which has one of the best understood circadian clock systems, offers a powerful experimentally-accessible system for exploring the clock mechanism at molecular levels. Like circadian oscillators in the higher eukaryotic organisms, the Neurospora oscillator consists of an auto regulatory negative feedback loop. In this negative feedback loop, two WHITE COLLAR proteins (WC-1 and WC-2) are the positive elements that form a WC complex that activate the transcription of the frequency (frq) gene. The negative elements are FRQ and FRH, a FRQ-interacting RNA helicase, which form a complex that inhibits the WCC activity. As the Period proteins in animal clock systems, FRQ is progressively phosphorylated and its phosphorylation promotes its degradation via the ubiquitin-proteasome pathway mediated by FWD-1, a F- box/WD40 containing protein which is the homolog of the mammalian beta-TRCP. FRQ stability is a major determinant in circadian period length. In Specific Aim 1, we will determine how phosphorylation-dependent FRQ degradation defines the circadian period of the clock. We will determine the effect of multiple FRQ phosphorylation on its interaction with FWD-1, will reconstitute the phosphorylation-dependent FRQ ubiquitination in vitro, and understand how FRQ phosphorylation is regulated different kinases. In Specific Aim2, we will determine the role and mechanism of the nuclear-cytoplasmic shuttling of the clock proteins and understand how their cellular localization is regulated. In Specific Aim3, we will determine the mechanism of a novel regulation that controls FRQ expression and function. These related but independent objectives will help us to elucidate the mechanism of the Neurospora clock in genetic, biochemical, and molecular terms. Because of the similarities between the Neurospora and animal clocks, our results will provide important information on how eukaryotic circadian clock functions. PUBLIC HEALTH RELEVANCE: Circadian rhythms are daily endogenous oscillations of biochemical, cellular, developmental, and behavioral activities observed in virtually all organisms. The molecular machinery that permits the measurement of time is referred to as the biological clock or circadian clock and its output as circadian rhythms. The importance of biological clocks in human physiology and mental health is evident from their ubiquitous influence on a wide range of cellular and organismal processes, including sleep/wake and body temperature cycles, endocrine functions, drug tolerance and resistance, and the phenomenon of jet lag. The malfunction of the clock is known to be associated with several forms of human psychiatric illness and with sleep disorders. A better understanding of circadian clocks will potentially lead to new therapeutic approaches for treating human diseases.